标题: | 探讨葡萄糖浓度对中国大仓鼠卵巢细胞增生之研究 Mechanism Involved in Inhibition of High Concentration of Glucose on The Proliferation of Chinese Hamster Ovary Cells |
作者: | 俞铭诚 Ming-Chen Yu 曾庆平 Ching-Ping Tseng 生物科技学系 |
关键字: | 中国大仓鼠卵巢细胞;葡萄糖;细胞增生;Chinese Hamster Ovary (CHO) cell;glucose;cAMP;Mitogen-activated Protein kinase;cAMP dependent protein kinase A;Proliferation |
公开日期: | 2000 |
摘要: | 本实验室先前研究发现CHO细胞株在高葡萄糖浓度(10 g/L)下,葡萄糖消耗速率会增加2倍,乳酸累积速率增加30 %,而生长倍增时间由13小时延长为18小时,并且细胞内cAMP含量增加2∼3倍。在人类血管内皮细胞亦发现在高葡萄糖浓度下,细胞倍增时间与细胞内cAMP皆有增加之趋势。由于细胞内之cAMP为二级讯息传导物,对细胞内酵素活性、细胞分化及增生等均扮演重要角色。由体外培养之肝细胞及人类乳癌细胞中发现,当提高细胞内cAMP含量,会调控MAP kinase磷酸化,进而抑制细胞增生,且MAP kinase活性和细胞分化及增生亦有关。 为了解此高葡萄糖浓度抑制是否受不同浓度血清之影响,我们利用低血清(2 %)培养CHO细胞株,来观察高及低(1 g/L)浓度葡萄糖在低血清浓度下对其生理影响,结果我们发现在高葡萄糖浓度环境下,细胞对葡萄糖消耗速率增加2倍、乳酸累积速率增加50 %、细胞内cAMP含量增加约3倍,此结果和高血清(10 %)培养条件下有相近结果,因此cAMP变化主要受葡萄糖浓度影响。由于在高葡萄糖浓度下,我们发现MAP kinase活性有上升现象,但加入PKA(cAMP dependent Protein kinase A) 抑制剂(H-8)后,MAP kinase活性则与低葡萄糖浓度相同;此外,亦观察不同细胞周期下葡萄糖对cAMP之影响,结果发现高葡萄糖浓度下,细胞内cAMP含量皆高于低葡萄糖浓所培养之细胞,且在G1时期表现最高,而MAP kinase活性在高葡萄糖浓度下则有延长现象。综合以上实验结果我们推论,葡萄糖可能经由cAMP活化MAP kinase或抑制 dephosphorylation,继而延长细胞增生时间。进一步实验证实在加入PKA抑制剂(H-8)后,高葡萄糖浓度之细胞生长速率回复至1.3/day,此结果与低葡萄糖浓度之细胞生长速率一致,因此本篇论文推论高葡萄糖浓度对细胞生长速率之抑制是经由cAMP调控,进而活化MAP kinase所影响。 The high glucose concentration inhibits Chinese Hamster Ovary (CHO) cell growth but the mechanism is still unclear. In previous studies, we found that the cell growth rate decreased from 1.3/day to 0.9/day when cells were grew in 10 g/L glucose concentration. In addition, the glucose consumption rate, lactate production rate, and the intracellular cAMP level increased twofold, 30 %, and threefold respectively. In this study, we have further demonstrated that the inhibition of the CHO cell growth was directly affected by high glucose concentration because it happened at both of high and low serum concentrations medium. The intracellular cAMP level regulates cell proliferation at high glucose growth condition in fibroblast cells. Therefore, the increase of cAMP might mediate the activation of Mitogen-activated Protein (MAP) kinase activity to inhibition of the CHO cells’ growth. In this study, the results showed that the MAP kinase phosphorylation did increase at high glucose concentration condition, and this activation was returned to the same level as grew in low glucose concentration when the cAMP signal was blocked. The high glucose concentration also regulated the CHO cell’s cell cycle during the G1 phase (0 to 8 hr growth). The intracellular cAMP level was increased and the phosphorylation of MAP kinase was postponed (or the dephosphorylation activity was inhibited) when glucose concentration was increased from 1 g/L to 10 g/L. Interestingly, the intracellular cAMP signals were blocked by cAMP dependent protein kinase A (PKA) inhibitor, and the MAP kinase phosphorylation activity as well as cell growth rate were returned to the same level as in low glucose concentration. Therefore, the results of these studies suggested that the high glucose might activate the MAP kinase activity by elevation of the intracellular cAMP level to inhibit the CHO cell growth. |
URI: | http://140.113.39.130/cdrfb3/record/nctu/#NT890111023 http://hdl.handle.net/11536/66571 |
显示于类别: | Thesis |